R E S E A R C H Open AccessInhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces leukocyte adhesion within the intestinal microcirculation in experimental endotox
Trang 1R E S E A R C H Open Access
Inhibition of lectin-like oxidized low-density
lipoprotein receptor-1 reduces leukocyte
adhesion within the intestinal microcirculation
in experimental endotoxemia in rats
Martin Landsberger1,2, Juan Zhou3, Sebastian Wilk4, Corinna Thaumüller4, Dragan Pavlovic4, Marion Otto1,
Sara Whynot3, Orlando Hung3, Michael F Murphy3, Vladimir Cerny3,5, Stephan B Felix1,2, Christian Lehmann3,4*
Abstract
Introduction: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major endothelial receptor for oxidized low-density lipoprotein, is also involved in leukocyte recruitment Systemic leukocyte activation in sepsis represents a crucial factor in the impairment of the microcirculation of different tissues, causing multiple organ failure and subsequently death The aim of our experimental study was to evaluate the effects of LOX-1 inhibition
on the endotoxin-induced leukocyte adherence and capillary perfusion within the intestinal microcirculation by using intravital microscopy (IVM)
Methods: We used 40 male Lewis rats for the experiments Ten placebo-treated animals served as a control Thirty animals received 5 mg/kg lipopolysaccharide (LPS) intravenously Ten endotoxemic rats remained untreated In 10 LPS animals, we administered additionally 10 mg/kg LOX-1 antibodies Ten further LPS animals received a
nonspecific immunoglobulin (rat IgG) intravenously After 2 hours of observation, intestinal microcirculation was evaluated by using IVM; the plasma levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-a) were determined; and LOX-1 expression was quantified in intestinal tissue with Western blot and reverse-transcription polymerase chain reaction (PCR)
Results: LOX-1 inhibition significantly reduced LPS-induced leukocyte adhesion in intestinal submucosal venules (P < 0.05) At the protein and mRNA levels, LOX-1 expression was significantly increased in untreated LPS animals (P < 0.05), whereas in animals treated with LOX-1 antibody, expression of LOX-1 was reduced (P < 0.05) MCP-1 plasma level was reduced after LOX-1 antibody administration
Conclusions: Inhibition of LOX-1 reduced leukocyte activation in experimental endotoxemia LOX-1 represents a novel target for the modulation of the inflammatory response within the microcirculation in sepsis
Introduction
Sepsis, severe sepsis, and septic shock are attributed
with a high incidence and mortality in critically ill
patients [1] The development of septic multiple organ
failure is linked to the impairment of the
microcircula-tion of vital and nonvital organs Several factors
contri-bute to the impairment of the microcirculation in sepsis,
including disseminated intravascular coagulation, capil-lary leakage, and leukocyte adhesion and infiltration [2] LOX-1 is a 50-kDa type II membrane protein that structurally belongs to the C-type lectin family, with a short intracellular N-terminal hydrophilic and a long extracellular C-terminal hydrophilic domain separated
by a hydrophobic domain of 26 amino acids [3] Infor-mation concerning the pathophysiologic role of LOX-1
is accumulating The unique lectin-like structure enables LOX-1 to recognize a wide range of negatively charged substances, including oxidized low-density lipoproteins
* Correspondence: chlehmann@dal.ca
3
Department of Anesthesia, Dalhousie University, 1276 South Park St., Halifax,
NS, B3 H 2Y9, Canada
Full list of author information is available at the end of the article
© 2010 Lehmann et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
Trang 2(OxLDLs), damaged or apoptotic cells, (endo)toxins, and
pathogenic microorganisms [3] After binding to LOX-1,
these ligands can either be internalized by endocytosis
or phagocytosis or can remain at the cell surface for
adhesion Under physiologic conditions, LOX-1 may
serve to clean up cellular debris and other related
mate-rials, and it might play a role in host defense [4-6] In
pathologic states, LOX-1 might be involved in the
bind-ing of OxLDL and cellular ligands to activate endothelial
cells, the transformation of smooth muscle cells (SMCs),
and the accumulation of lipids in macrophages,
espe-cially important in the development of atherosclerosis
[7-9] The expression of LOX-1 is induced by stimuli as
rapidly as other kinds of cell-adhesion molecules and
selectins, suggesting that LOX-1 belongs to the so-called
class of immediate-early genes [10] LOX-1 is a potent
mediator of ‘’endothelial dysfunction’’: binding of
endothelial LOX-1 by ligands induces superoxide
gen-eration, inhibits nitric oxide production, enhances
endothelial adhesiveness for leukocytes, and induces
expression of chemokines [11-13]
In a rat model with endotoxin-induced uveitis, an
antibody against LOX-1 suppressed leukocyte infiltration
and protein exudation [10] However, the effects of
LOX-1 inhibition on leukocyte activation during
sys-temic inflammation must be further elucidated
The intestinal microcirculation is crucial in the
patho-genesis of septic multiple organ failure [2] Therefore,
the aim of our experimental study was to evaluate the
effects of LOX-1 inhibition on endotoxin-induced
leuko-cyte adherence and the impaired capillary perfusion in
the intestinal microcirculation during experimental
endotoxemia by using intravital microscopy (IVM)
Materials and methods
Animals
The study was performed in accordance with
interna-tionally recognized guidelines, the local Instructions for
Animal Care of the University of Greifswald, and the
German Law on the Protection of Animals (approved by
the Landesamt für Landwirtschaft,
Lebensmittelsicher-heit und Fischerei Mecklenburg-Vorpommern) Forty
male Lewis rats (200 to 250 g) were obtained from
Charles River Laboratories (Sulzfeld, Germany) and kept
under constant conditions of a 12-hour light/dark cycle
at 25°C with a humidity of 55% After the experiments,
the animals were sacrificed by using a pentobarbital
overdose
Anesthesia and preparation
Anesthesia was induced by intraperitoneal injection of a
bolus of 60-mg/kg pentobarbital (Synopharm GmbH &
Co KG, Barsbüttel, Germany) To maintain an adequate
depth of anesthesia, the animals received 5 mg/kg
pentobarbital intravenously every hour For preparation, the animals were placed in a supine position, and a straight skin incision from the chin to the sternum was made The polyethylene catheters (PE 50; internal dia-meter, 0.58 mm; external diadia-meter, 0.96 mm; Portex; Smiths Medical, Hythe, Kent, UK) were introduced into the left external jugular vein and common carotid artery The intraarterial catheter provided a continuous monitoring of mean arterial blood pressure (MAP) and heart rate (HR) (monitor: Philips LDH 2106/00; Philips, Eindhoven, The Netherlands) To secure the airway, a trimmed venous catheter (16 G, BD Insyte-W; Becton Dickinson GmbH, Germany) was introduced into the trachea via tracheotomy The animals breathed sponta-neously in room air To maintain a constant body tem-perature of 37°C ± 0.5°C, the animals were placed on an electric blanket To expose the intestine, a median lapar-otomy subsequently was performed from the xyphoid to the symphysis
Protocol
We administrated 5 mg/kg lipopolysaccharide (LPS) from Escherichia coli, serotype O157:H7 (Sigma-Aldrich Chemie, Steinheim, Germany) intravenously in
30 animals Fifteen minutes after LPS administration,
10 of the animals received 10 mg/kg LOX-1 antibody (LPS/Anti-LOX group) intravenously To differentiate specific LOX-1 effects from unspecific antibody effects, another 10 animals received rat immunoglobulin G (LPS/IgG group) The remaining 10 animals did not receive any treatment (LPS group) The control group (CON) animals received an equivalent volume of pla-cebo (normal saline; Delta Select GmbH, Dreieich, Germany)
Intravital microscopy was performed 2 hours after LPS administration Blood samples for the laboratory ana-lyses were drawn 30 and 120 minutes after the start of the experiments At the end of the IVM experiments, animals were sacrificed, and samples of intestinal tissue taken for further protein and mRNA analysis
Intravital fluorescence microscopy
A part of the intestine approximately 5 cm proximal to the ileocecal valve was identified and placed on an adjustable object table on the microscope The config-uration and procedure for IVM were described pre-viously [14] In brief, leukocytes were stained in vivo by
an intravenous injection of 0.2 ml 0.05% Rhodamine 6G solution (Sigma-Aldrich Chemie GmbH, Steinheim, Ger-many) Capillary perfusion was made visible by the administration of 5% FITC-albumin solution (1 ml/kg, intravenous; Sigma-Aldrich Chemie) For evaluation of the leukocyte adhesion, the intestinal section was focused at the submucosal level Six visual fields
Trang 3containing nonbranching, collecting venules (V1) over a
length of at least 300 μm, as well as another six visual
fields revealing similar postcapillary venules (V3), were
observed and recorded for 35 seconds each To obtain
comparable results, we selected vessels of comparable
size (V1, 60 to 80μm; V3, 30 to 40 μm) The same
pro-cedure was done by focusing random fields of the
capil-laries within the longitudinal as well as the circular
muscle layer and the mucosa Evaluation of all the video
sequences was accomplished after the experiments by
analyzing the videotapes off line with a
computer-con-nected video system and software (CapImage; Zeintl,
Heidelberg, Germany) Leukocyte adherence was defined
as the number of leukocytes that stayed immobile for at
least 30 seconds on an oblique, cylindrical endothelial
surface (number/mm2) FCD was measured as the
length of capillaries with observable erythrocyte
perfu-sion in relation to a predetermined rectangular field
(cm/cm2)
Cloning of ratLOX-1 gene and preparation of
polyclonal antibodies
The coding region for the triple-repeat motive comprising
amino acids 94 to 232 of LOX-1 protein from rat
was amplified with the oligonucleotides 5
’-CGGGATC-CAAGAATCAAAGAGGGAACTGAA-3’ (5’-end) and
5’-CCGCTCGAGACCTGAAGAGTTTGCAGCTCT-3’
(3’-end), which introduced BamHI and XhoI restriction
sites (underlined) The BamHI/XhoI-fragment was then
fused in frame to the glutathion-S-transferase (GST) gene
into pGEX-5X-3 (GE Healthcare, Freiburg, Germany) to
obtain the plasmid pGEX-5X-3/AA94-232 The construct
was sequenced, and identity with the published LOX-1
sequence from rat was confirmed Recombinant GST/
AA94-232 protein was produced in Escherichia coli strain
BL21(DE3)pLysS, purified, and cleaved with Factor Xa for
16 hours AA9494-232 was used to prepare a polyclonal
antiserum in rabbits with a standard immunization
protocol [15]
Quantitative reverse transcription polymerase
chain reaction
Quantification of LOX-1 andb-actin, as an endogenous
housekeeping gene, mRNA expression was performed
by using mRNA Assays-on-Demand (Applera
Deutsch-land GmbH, Darmstadt, Germany) on an Applied
Bio-systems ABI Prism 7700, as described previously [16]
Protein isolation and quantification
At the end of the IVM experiments, animals were
sacri-ficed, and intestinal tissue was dissected, washed twice
with media, and homogenized in 10 mM Tris (pH 7.4,
1 mM sodium ortho-vanadate, and 1% (wt/vol) SDS)
Protein concentrations were measured by using the
bicinchoninic acid (BCA) Protein Assay Kit (Perbio Science, Bonn, Germany)
Laboratory analyses
Blood samples were drawn 30 and 120 minutes after start of the experiments Monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor-alpha (TNF-a) plasma levels were measured according to the manu-facturer’s instructions (FlowCytomix; Bender MedSys-tems, Vienna, Austria)
Statistical analyses
Results were analyzed by using the software Prism 5 (GraphPad Software, La Jolla, CA, USA) First, data were tested for normal distribution by using the Kolmogorov-Smirnov test If normal distribution was established, one-way analysis of variance (ANOVA) was performed
If significant differences appeared, a post hoc analysis with Dunn’s Multiple Comparison Test was conducted The investigations of values in multifactorial design were examined by means of two-way analysis of variance (two-way repeated-measures ANOVA) A value of
P < 0.05 was considered statistically significant
Results
The protocol was performed as outlined All animals survived the observation period and could be included
in the study
Microcirculation
We observed a significant increase of the number of adherent leukocytes in V1 (collecting) and V3 (postca-pillary) venules of untreated LPS animals compared with control animals (Figure 1a and 1b; P < 0.05) This increase was completely abolished in the LOX-1-anti-body-treated LPS group (P < 0.05) Unspecific immuno-globulin administration did not influence leukocyte adhesion in LPS-challenged animals Functional capillary density was not significantly impaired in these endotoxe-mia experiments (Table 1)
LOX-1 expression
Endotoxemia resulted in a significant increase in LOX-1 protein expression (Figure 2a) Administration of the antibody against LOX-1 significantly prevented the upregulation of LOX-1 protein expression Unspecific immunoglobulin had no effect on LOX-1 protein expression in the presence of LPS Effects of LPS and anti-LOX-1 were confirmed at the mRNA level by RT-PCR (Figure 2b)
MCP-1 and TNF-a release
MCP-1 plasma levels were significantly elevated in all endotoxemic groups (Figure 3a) MCP-1 release was
Trang 4significantly reduced in the LPS/Anti-LOX group in
comparison to untreated or IgG-treated LPS animals
TNF-a concentrations were increased in all
endotoxe-mic animals compared with those in the control group
(Figure 3b; P < 0.05)
Figure 1 Adherent leukocytes in V1 (a) and V3 (b) venules
( n/mm 2 ) CON, control group (n = 10); LPS, lipopolysaccharide
group (n = 10); LPS/Anti-LOX, lipopolysaccharide and
LOX-1-antibody group (n = 10); LPS/IgG, lipopolysaccharide and
unspecific immunoglobulin group (n = 10) #P < 0.05 vs CON;
§P < 0.05 vs LPS.
Table 1 Functional capillary density
CON LPS LPS/Anti-LOX LPS/IgG Longitudinal muscle layer (mm) 140.5 ± 21.7 160.0 ± 10.9 169.4 ± 10.7 152.2 ± 29.9 Circular muscle layer (mm) 90.7 ± 33.9 115.7 ± 41.8 131.7 ± 21.0 119.8 ± 37.6 Mucosa (mm) 471.2 ± 51.2 456.9 ± 65.4 507.4 ± 51.5 526.9 ± 45.1
CON, control group; LPS, lipopolysaccharide group (untreated); LPS/Anti-LOX, LPS animals treated with the LOX antibody; LPS/IgG, LPS animals treated with unspecific immunoglobulin G; functional capillary density, cm/cm 2
; values expressed as mean ± SD; n = 10 per group.
Figure 2 Expression of LOX-1 protein (a) and mRNA (b) in rat intestine Intestinal tissue was harvested from control (CON), lipopolysaccharide (LPS), lipopolysaccharide, LOX-1-antibody treated (LPS/Anti-LOX), and lipopolysaccharide and unspecific immunoglobulin treated (LPS/IgG) animals (n = 10 for each group) Total protein and mRNA were extracted from rat intestine tissue, and the expression of LOX-1 was evaluated with Western blot and reverse transcription PCR, respectively #P < 0.05 vs CON;
§P < 0.05 vs LPS.
Trang 5Mean arterial pressure (MAP, Figure 4a) and heart rate
(Figure 4b) were stable in control animals over the
2-hour period of the investigation Between 30 and
90 minutes, a significant decrease of MAP in all
endotoxe-mic groups was noted compared with that in the control
group A significant decrease of heart rate was observed in
the LPS-only group at 90 minutes, as compared with the
control group Endotoxemic groups plus treatment (either
LOX-1 antibody or unspecific immunoglobulin) showed a
significant increase in heart rate between 30 and 90
min-utes, which persisted for the duration of the experiments
Heart rates were also significantly higher in these groups
as compared with the LPS-only group
Discussion
Administration of antibodies against LOX-1 significantly
reduced endotoxin-induced leukocyte adherence in
intestinal submucosal venules LOX-1 expression was reduced significantly at both mRNA and protein levels
in animals treated with the antibody MCP-1 plasma levels were found to be decreased after administration
of antibodies against LOX-1
The exposure of LDL to oxidative stress generates OxLDL The expression of the OxLDL receptor LOX-1
is upregulated by the increased occurrence of OxLDL Interestingly, the OxLDL-induced upregulation is inhib-ited by antibodies against LOX-1 [17,18] Endotoxemia and sepsis are pathologic conditions with increased oxi-dative stress and release of reactive oxygen species (ROS) Our findings suggest that LOX-1 antibodies are also able to reduce the endotoxin-induced expression of LOX-1
Because ROS function as signal-transduction mole-cules that modulate the activity of the transcription
Figure 3 Plasma levels of MCP-1 (a) and TNF- a (b) CON, Control
group (n = 10); LPS, lipopolysaccharide group (n = 10);
LPS/Anti-LOX, lipopolysaccharide and LOX-1-antibody group (n = 10); LPS/
IgG, lipopolysaccharide and unspecific immunoglobulin group
(n = 10) #P < 0.05 vs CON; §P < 0.05 vs LPS.
Figure 4 Mean arterial pressure and heart rate CON, Control group (n = 10); LPS, lipopolysaccharide group (n = 10); LPS/Anti-LOX, lipopolysaccharide and LOX-1-antibody group (n = 10); LPS/ IgG, lipopolysaccharide and unspecific immunoglobulin group (n = 10); #P < 0.05 vs CON; §P < 0.05 vs LPS.
Trang 6factors, various changes via gene expression accompany
the changes in redox status of the cells Activation of
NF-B, a redox-sensitive transcription factor, induces
upregulation in the expression of vasoconstrictive
mole-cules, adhesion molemole-cules, and chemokines [19,20]
Actually, activation of LOX-1 in endothelial cells
induces the expression of endothelin-1, AT1 receptor,
E-selectin, P-selectins, VCAM-1 and ICAM-1 (30), and
MCP-1 [11] These gene products increase vascular
tonus and promote leukocyte-endothelial interactions
and the release of additional pro-inflammatory signals
We confirmed in our experiments that leukocyte
adhe-sion and MCP-1 levels can be influenced by LOX-1
inhibition in experimental endotoxemia in rats
TNF-a is an early proinflammatory cytokine Kume
et al [21] showed that TNF-a increases cell-surface
expression of LOX-1 in a concentration-dependent
manner, and peak levels of LOX-1 expression are at
8 to 12 hours with continuous TNF-a stimulation
in vitro TNF-a appeared to activate the transcription of
LOX-1, as measured by nuclear run-off assay Time to
peak concentrations of TNF-a has been suggested to be
1 hour after endotoxin challenge [22] In our
experi-ments, TNF-a was measured about 2 hours after
endo-toxin challenge This may explain why we did not
observe differences in the TNF-a levels between the
experimental groups
OxLDL itself has also been well known to play a key
role in the adherence of monocytes to the activated
endothelium Possible intracellular processes include
activation of protein kinase C, mitogen-activated protein
kinase (MAPK), and the subsequent upregulation of
MCP-1 [12,23,24] Li et al [11] found that incubation of
endothelial cells with Ox-LDL increased the
phosphory-lation of MAPK In these experiments, OxLDL also
upregulated MCP-1 expression (protein and mRNA)
and monocyte adhesion to the endothelial cells through
activation of LOX-1 In a model of low-dose
endotoxin-induced uveitis, antibodies against LOX-1 efficiently
suppressed leukocyte infiltration and protein exudation
In situ videomicroscopic analyses of leukocyte
interac-tions with retinal veins revealed that LOX-1
anti-body reduced the number of rolling leukocytes and
increased the velocity of rolling, suggesting that LOX-1
functions as a vascular tethering ligand The ability of
LOX-1 to capture leukocytes under physiologic shear
was confirmed in an in vitro flow model [10] We also
were able to show that endotoxin-induced leukocyte
adhesion can be influenced by anti-LOX-1
administra-tion Leukocyte adhesion was completely abolished in
the LOX-1 antibody-treated LPS group in rats
Influences of a reduced perfusion pressure, the typical
response to endotoxemia, on the findings within the
microcirculation cannot be excluded completely, but the
reduction in mean arterial blood pressure in our experi-ments was only temporary and still in a physiologic range At the time of the evaluation of the microcircula-tion, perfusion pressure in endotoxemic animals was not significantly different from that of controls Further-more, capillary perfusion, as measured by the functional capillary density, was unchanged in endotoxemic ani-mals, also indicating a negligible impact of the perfusion pressure in our experiments Several studies observed a significant impairment of functional capillary density during experimental endotoxemia [25-27] However, the extent of the impairment of the functional density depends on several factors (for example, the serotype, the dosage, and the endotoxin activity of the LPS used for the induction of endotoxemia and the organ/tissue studied for changes in the microcirculation) It was interesting to observe that FCD response and leukocyte activation can be dissociated We interpret the missing effect of endotoxemia on the FCD in our experimental study as associated with the low severity of the model (no septic shock)
Reduction of leukocyte adhesion and impact on func-tional capillary density and cytokine response, as well as the effect on survival by administration of LOX-1 anti-bodies in endotoxemia, should be studied in further ani-mal experiments These studies will verify the potential use of this therapeutic approach in a clinical setting
Conclusions
Inhibition of the lectin-like oxidized low-density lipopro-tein receptor-1 resulted in a reduction of endotoxin-induced intestinal leukocyte adhesion Therefore, this receptor may represent a novel target for the modula-tion of the inflammatory response within the microcir-culation in sepsis
Key messages
• Lectin-like oxidized low-density lipoprotein recep-tor-1 (LOX-1) is involved in leukocyte recruitment
• Systemic leukocyte activation represents a crucial factor in the pathogenesis of sepsis
• Inhibition of LOX-1 reduced leukocyte activation
in experimental endotoxemia
• In rats treated with LOX-1 antibody, expression of LOX-1 was reduced
• LOX-1 represents a novel target for the modula-tion of the inflammatory response within the micro-circulation in sepsis
Abbreviations ELISA: enzyme-linked immunosorbent assay; FCD: functional capillary density; FITC: fluorescein isothiocyanate; HR: heart rate; IgG: immunoglobulin G; IL: interleukin; IVM: intravital fluorescence microscopy; LDL: low-density lipoprotein; LOX-1: lectin-like oxidized low-density lipoprotein receptor-1;
Trang 7LPS: lipopolysaccharide; MAP: mean arterial pressure; MCP-1: monocyte
chemoattractant protein-1; OxLDL: oxidized low-density lipoprotein; ROS:
reactive oxygen species; RT-PCR: reverse transcription polymerase chain
reaction; SMC: smooth muscle cell; TNF- α: tumor necrosis factor-alpha; V1:
collecting venule; V3: postcapillary venule.
Acknowledgements
The authors thank R Dressler and S Will for excellent technical assistance.
Author details
1 Department of Internal Medicine B, University Hospital Greifswald,
Friedrich-Loeffler-Strasse 23 a, D-17475 Greifswald, Germany 2 Research Center of
Pharmacology and Experimental Therapeutics, University Hospital Greifswald,
Friedrich-Loeffler-Strasse 23 d, D-17475 Greifswald, Germany.3Department of
Anesthesia, Dalhousie University, 1276 South Park St., Halifax, NS, B3 H 2Y9,
Canada.4Department of Anesthesiology and Intensive Care Medicine,
University Hospital Greifswald, Friedrich-Loeffler-Strasse 23a, D-17475
Greifswald, Germany.5Department of Anesthesiology and Intensive Care
Medicine, University Hospital Hradec Kralove, Charles University in Prague,
Sokolska 581, 500 05 Hradec Kralove, Czech Republic.
Authors ’ contributions
ML and MO performed cloning, expression, and antibody preparation of
LOX-1, Western blot, and RT-PCR analysis SW and CT carried out intravital
microscopy ML and CL conceived of the study, analyzed data, and drafted
the manuscript DP, MM, and VC made substantial contributions to the
conception and design of the study, and DP supervised the IVM
experimental procedure SBF, JZ, SW, and OH have been involved in revising
the manuscript critically for important intellectual content All authors read
and approved the final manuscript.
Competing interests
Parts of this work were supported by a grant from the Department of
Cardiovascular Medicine within the NBL3 program (reference 01 ZZ 0403) of
the German Federal Ministry of Education and Research (to ML and SBF).
Supported in part by Research project MZO 00179906 from the University
Hospital Hradec Kralove, Czech Republic (to VC).
Received: 30 April 2010 Revised: 3 August 2010
Accepted: 10 December 2010 Published: 10 December 2010
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doi:10.1186/cc9367
Cite this article as: Landsberger et al.: Inhibition of lectin-like oxidized
low-density lipoprotein receptor-1 reduces leukocyte adhesion within
the intestinal microcirculation in experimental endotoxemia in rats.
Critical Care 2010 14:R223.
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